IGEM:Groningen/Notebook/iGEM 2011/2011/09/09: Difference between revisions
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== | ==9-9-11== | ||
<br> Picked up a new Plasmid DNA isolation kit! :) | |||
<br> Strains for FACS measurements grew!:) | |||
<br> Stickers for sequencing finally arrived!:) | |||
<br> | |||
<br> Colony PCR of PBAD-cI-LVA-DT | |||
<br> | |||
<br> The conventional taq polymerase tube was empty:( So I borrowed the dreamtaq from MolGen. For more infon about | |||
<br> dreamtaq:http://www.fermentas.com/en/products/all/modifying-enzymes/thermophilic-polymerases/ep070 | |||
<br> Addition of MgCl2 in the mastermix will not be necessary, since the Dreamtaq buffer already contains MgCl2. | |||
<br> For the rest, the normal taq mastermix scheme can be used, but the amount of microliters normally used to add MgCl2 need to be | |||
<br> | |||
<br> Taq 10× buffer: 40μl | |||
<br> dNTPs 10mM: 8μl | |||
<br> Forward Biobrick primer: 8μl | |||
<br> Reverse Biobrick primer: 8μl | |||
<br> Taq DNA polymerase: 2μl | |||
<br> MQ water: 334μl | |||
<br> | |||
<br> PCR conditions: | |||
<br> Preheated lid: 111°C | |||
<br> Denaturation: 94°C for 10 min. | |||
<br> Cycle 33×: | |||
<br> denaturation: 94°C for 30s. | |||
<br> annealing: 60°C for 30s. | |||
<br> Extension: 72°C for 2,5 min. | |||
<br> Final extension: 72°C for 10 min. | |||
<br> Store infinite at 4°C | |||
<br> | |||
<br> Analyse on a 1% TBE agarose gel. | |||
<br> | |||
<br> Send samples for sequencing! | |||
<br> | |||
<br> | |||
Revision as of 02:17, 9 September 2011
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